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Delta-like 4 is indispensable in thymic environment specific for T cell development.

Hozumi K, Mailhos C, Negishi N, Hirano K, Yahata T, Ando K, Zuklys S, Holländer GA, Shima DT, Habu S - J. Exp. Med. (2008)

Bottom Line: Further analysis showed that the double-negative cell fraction was lacking T cell progenitors.The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4.These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Research Center for Embryogenesis and Organogenesis, Tokai University School of Medicine, Isehara 259-1193, Japan. hozumi@is.icc.u-tokai.ac.jp

ABSTRACT
The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

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Specific abrogation of Dll4 on thymic epithelium. (A) Targeted insertion of loxP sequences flanking a part of exon 1 and whole exons 2 and 3 of the Dll4 gene. Numbers indicate exon number. 1*, first exon modified with loxP sequence; 1**, part of exon 1 after gene deletion; ATG, translational initiation codon of Dll4; triangles, loxP sequences; open boxes, Dll4 exons; B, BglII; C, ClaI; RI, EcoRI; Xb, XbaI; Xh, XhoI. (B) Cre activity was targeted to thymic epithelia using mice, designated FoxN1-Cre, in which this recombinase is under the transcriptional control of the FoxN1 locus. The timing and specificity of Cre-mediated recombination was visualized by the expression of enhanced GFP (eGFP) in thymus tissue sections of E12.5 (left) and 6-wk-old (right) crosses of FoxN1-Cre mice with the transgenic Z/EG reporter mice. The thymus anlage in the left image is outlined with a dashed line. C, cortex; M, medulla. (C) The expression of Dll4 on thymic epithelial, but not endothelial, cells was abrogated in Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) mice. Epithelial and endothelial cells in the thymus were identified by the expression of cytokeratin (CK, green, left and middle) and CD31 (red, right), respectively, with Dll4 (red, middle; green, right). Bars: (B and C) 50 μm.
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fig2: Specific abrogation of Dll4 on thymic epithelium. (A) Targeted insertion of loxP sequences flanking a part of exon 1 and whole exons 2 and 3 of the Dll4 gene. Numbers indicate exon number. 1*, first exon modified with loxP sequence; 1**, part of exon 1 after gene deletion; ATG, translational initiation codon of Dll4; triangles, loxP sequences; open boxes, Dll4 exons; B, BglII; C, ClaI; RI, EcoRI; Xb, XbaI; Xh, XhoI. (B) Cre activity was targeted to thymic epithelia using mice, designated FoxN1-Cre, in which this recombinase is under the transcriptional control of the FoxN1 locus. The timing and specificity of Cre-mediated recombination was visualized by the expression of enhanced GFP (eGFP) in thymus tissue sections of E12.5 (left) and 6-wk-old (right) crosses of FoxN1-Cre mice with the transgenic Z/EG reporter mice. The thymus anlage in the left image is outlined with a dashed line. C, cortex; M, medulla. (C) The expression of Dll4 on thymic epithelial, but not endothelial, cells was abrogated in Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) mice. Epithelial and endothelial cells in the thymus were identified by the expression of cytokeratin (CK, green, left and middle) and CD31 (red, right), respectively, with Dll4 (red, middle; green, right). Bars: (B and C) 50 μm.

Mentions: Dll4 is the third member of the Dll family characteristically expressed in the endothelium and thymus (8–10, 15). Immunohistochemical analysis with Dll4-specific antibody revealed that Dll4 was dominantly expressed on the thymic epithelium with cytokeratin (Fig. 1 A), especially in the cortex (K8+K5− region), which is where early T cell development occurs (Fig. 1 B). In contrast, it was found on endothelial cells, but not on the epithelium in gut or salivary gland (Fig. 1 C). These findings suggested that the distinctive expression of Dll4 on the thymic epithelium contributes to the formation of a thymus-specific environment. To examine the physiological significance of Dll4 on the thymic epithelium for T cell development, Dll4-targeting mice, with the Dll4 gene flanked by a loxP sequence (designated Dll4-floxed), were established (Fig. 2 A). The floxed allele of Dll4 gene was removed by Cre recombinase, resulting in the loss of the translational initiation site. These mice were bred with FoxN1-Cre mice, where the Cre gene is driven by the regulatory element of FoxN1 gene (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20080134/DC1). The specific activity of Cre recombinase in FoxN1-Cre mice was observed as the ectopic expression of GFP in the thymic epithelium by crossing with the reporter transgenic mice (16) at the embryonic or adult stage (Fig. 2 B). In mice homozygous for floxed Dll4 with FoxN1-Cre transgene (FoxN1Cre:Dll4-floxed), the expression of Dll4 was completely abrogated on TECs (Fig. 2 C, bottom middle), but was still intact on endothelial cells bearing CD31 (Fig. 2 C, bottom right). These results indicated that the gene deletion was specifically induced in epithelial cells in the thymus. Moreover, this deletion of the Dll4 gene did not affect the expression of Dll1 in the thymus (Fig. S2). Interestingly, cortical and medullary regions were almost indistinguishable in the thymus of mutant mice (unpublished data).


Delta-like 4 is indispensable in thymic environment specific for T cell development.

Hozumi K, Mailhos C, Negishi N, Hirano K, Yahata T, Ando K, Zuklys S, Holländer GA, Shima DT, Habu S - J. Exp. Med. (2008)

Specific abrogation of Dll4 on thymic epithelium. (A) Targeted insertion of loxP sequences flanking a part of exon 1 and whole exons 2 and 3 of the Dll4 gene. Numbers indicate exon number. 1*, first exon modified with loxP sequence; 1**, part of exon 1 after gene deletion; ATG, translational initiation codon of Dll4; triangles, loxP sequences; open boxes, Dll4 exons; B, BglII; C, ClaI; RI, EcoRI; Xb, XbaI; Xh, XhoI. (B) Cre activity was targeted to thymic epithelia using mice, designated FoxN1-Cre, in which this recombinase is under the transcriptional control of the FoxN1 locus. The timing and specificity of Cre-mediated recombination was visualized by the expression of enhanced GFP (eGFP) in thymus tissue sections of E12.5 (left) and 6-wk-old (right) crosses of FoxN1-Cre mice with the transgenic Z/EG reporter mice. The thymus anlage in the left image is outlined with a dashed line. C, cortex; M, medulla. (C) The expression of Dll4 on thymic epithelial, but not endothelial, cells was abrogated in Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) mice. Epithelial and endothelial cells in the thymus were identified by the expression of cytokeratin (CK, green, left and middle) and CD31 (red, right), respectively, with Dll4 (red, middle; green, right). Bars: (B and C) 50 μm.
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fig2: Specific abrogation of Dll4 on thymic epithelium. (A) Targeted insertion of loxP sequences flanking a part of exon 1 and whole exons 2 and 3 of the Dll4 gene. Numbers indicate exon number. 1*, first exon modified with loxP sequence; 1**, part of exon 1 after gene deletion; ATG, translational initiation codon of Dll4; triangles, loxP sequences; open boxes, Dll4 exons; B, BglII; C, ClaI; RI, EcoRI; Xb, XbaI; Xh, XhoI. (B) Cre activity was targeted to thymic epithelia using mice, designated FoxN1-Cre, in which this recombinase is under the transcriptional control of the FoxN1 locus. The timing and specificity of Cre-mediated recombination was visualized by the expression of enhanced GFP (eGFP) in thymus tissue sections of E12.5 (left) and 6-wk-old (right) crosses of FoxN1-Cre mice with the transgenic Z/EG reporter mice. The thymus anlage in the left image is outlined with a dashed line. C, cortex; M, medulla. (C) The expression of Dll4 on thymic epithelial, but not endothelial, cells was abrogated in Dll4lox/loxFoxN1-Cre (FoxN1Cre:Dll4-floxed) mice. Epithelial and endothelial cells in the thymus were identified by the expression of cytokeratin (CK, green, left and middle) and CD31 (red, right), respectively, with Dll4 (red, middle; green, right). Bars: (B and C) 50 μm.
Mentions: Dll4 is the third member of the Dll family characteristically expressed in the endothelium and thymus (8–10, 15). Immunohistochemical analysis with Dll4-specific antibody revealed that Dll4 was dominantly expressed on the thymic epithelium with cytokeratin (Fig. 1 A), especially in the cortex (K8+K5− region), which is where early T cell development occurs (Fig. 1 B). In contrast, it was found on endothelial cells, but not on the epithelium in gut or salivary gland (Fig. 1 C). These findings suggested that the distinctive expression of Dll4 on the thymic epithelium contributes to the formation of a thymus-specific environment. To examine the physiological significance of Dll4 on the thymic epithelium for T cell development, Dll4-targeting mice, with the Dll4 gene flanked by a loxP sequence (designated Dll4-floxed), were established (Fig. 2 A). The floxed allele of Dll4 gene was removed by Cre recombinase, resulting in the loss of the translational initiation site. These mice were bred with FoxN1-Cre mice, where the Cre gene is driven by the regulatory element of FoxN1 gene (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20080134/DC1). The specific activity of Cre recombinase in FoxN1-Cre mice was observed as the ectopic expression of GFP in the thymic epithelium by crossing with the reporter transgenic mice (16) at the embryonic or adult stage (Fig. 2 B). In mice homozygous for floxed Dll4 with FoxN1-Cre transgene (FoxN1Cre:Dll4-floxed), the expression of Dll4 was completely abrogated on TECs (Fig. 2 C, bottom middle), but was still intact on endothelial cells bearing CD31 (Fig. 2 C, bottom right). These results indicated that the gene deletion was specifically induced in epithelial cells in the thymus. Moreover, this deletion of the Dll4 gene did not affect the expression of Dll1 in the thymus (Fig. S2). Interestingly, cortical and medullary regions were almost indistinguishable in the thymus of mutant mice (unpublished data).

Bottom Line: Further analysis showed that the double-negative cell fraction was lacking T cell progenitors.The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4.These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Research Center for Embryogenesis and Organogenesis, Tokai University School of Medicine, Isehara 259-1193, Japan. hozumi@is.icc.u-tokai.ac.jp

ABSTRACT
The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

Show MeSH
Related in: MedlinePlus